An analysis of the underlying biochemical and genetic mechanisms that control gender and fertility in the Kuruma shrimp, Marsupenaeus japonicus (Bate)

Sellars, Melony J. (2007). An analysis of the underlying biochemical and genetic mechanisms that control gender and fertility in the Kuruma shrimp, Marsupenaeus japonicus (Bate)PhD Thesis, School of Integrative Biology, The University of Queensland.

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Centuries of experience with terrestrial agriculture have demonstrated the value of domestication and selective breeding to improve production efficiency, profitability and sustainability. Compared to terrestrial agriculture there are remarkably few aquaculture industries that have progressed beyond the use of wild or unselected genotypes. This presents a major opportunity for the global aquaculture industry to capitalize on the benefits of domestication and selective breeding. However, there is a parallel need to ensure that a balance is achieved between optimizing genetic gains and conserving the biodiversity of the captive and wild stocks from which they are derived. In progressing towards domestication and genetic improvement, one of the key areas of interest includes the factors that control gender and fertility. Gender is of particular interest in species that are sexually dimorphic, with one gender being more attractive from a production point of view. Fertility is of broad interest as a key aspect of reproductive performance. Controlling fertility is also one method of protecting unwanted or unlicensed breeding of elite genotypes and preventing escapees from mating with wild stocks. For most aquacuculture species there is a fundamental lack of knowledge of the underlying biochemical and genetic mechanisms that control gender and fertility. The focus of this study was on developing methods for controlling gender and fertility in the Kuruma shrimp Marsupenaeus japonicus (Bate). This high value species has been farmed for several decades using the progeny of wild broodstock and, more recently, progeny of domesticated, selectively bred captive broodstock. The biological, biochemical and genetic processes that control gender and fertility in M. japonicus are of direct commercial interest and of wider interest to science. Commercial interest is for two reasons: First, M. japonicus are sexually dimorphic with females growing 30% larger than males and second, recent success in the genetic improvement of M. japonicus has prompted farmers to seek ways to protect superior genetic stocks from unlicensed breeding and to prevent escapees from genetically contributing to natural fishery populations. Thus, the knowledge and technology that could permit farmers to cost-effectively produce all-female, sterile stocks could be of significant commercial value. More broadly, research directed towards this goal could provide new insights into the underlying biological, genetic and biochemical processes that control gender and reproduction in penaeid shrimp. Previous studies on inducing sterility have demonstrated that this can result in altered gende r ratios in the target species. Accordingly, the approach adopted in this study was to investigate these traits simultaneously. There were three principal research components in this project, each of which focused on a different approach to achieving gender and/or fertility control in M. japonicus. The three components were; (1) the potential of ploidy manipulation to control gender and fertility; (2) the effect of ionizing radiation on fertility; (3) the potential of genetic engineering to control gender determination and germ cell specification. PLOIDY MANIPULATION Previous research on M. japonicus has shown that triploids produced by preventing polar body (PB) II extrusion are always female and sterile, however, induction rates never result in 100% triploid progeny. Therefore, experiments in the present study focused on developing a technique to produce triploids with a 100% induction rate. As mating of tetraploids and diploids is the only documented technique that has achieved 100% triploids in other cultured species, the present study investigated methods to induce tetraploidy in M. japonicus. First I attempted to prevent the first division in mitosis using temperature and chemical shocks. Second I attempted to inhibit extrusion of PBI and both PBI and II using chemical shocks only. I was successful at stopping the first mitotic division, preventing the extrusion of PBI and preventing the extrusion of both PBI and II in M. japonicus by applying different shocks at different times post-spawning for different durations. Tetraploid M. japonicus embryos were produced by stopping the first division in mitosis, however, they were not viable and did not hatch. The most suitable treatment regime for inducing tetraploidy, giving frequency (number of spawnings induced that result in some polyploids) and induction rate (number of individuals within a spawning induction which are tetraploid or triploid) equal importance, was a 36°C shock administered 23 min post-spawning for a 5 or 10 min duration. Preventing the extrusion of PBI and both PBI and II using 150 µM 6-dimethylaminopurine at 1 to 3 min post-spawning for either a 4 to 5 min duration (timed to stop PBI extrusion) or a 16 min duration (timed to stop both PBI and II extrusion) consistently resulted in the production of viable triploid M. japonicus embryos, which hatched into nauplii. No tetraploids were produced by preventing the extrusion of PBI or both PBI and II. This is the first report of successful PBI, and PBI and II prevention in shrimp, and production of triploids using the reported treatment regimes. These findings indicate that manipulation of the ploidy level may not be suitable for producing 100% all-female, sterile M. japonicus, as all tetraploids produced to-date are not viable and all known triploidy induction techniques never result in 100% triploid progeny. There may, however, be commercial value in utilising the PBI triploidy induction methods of the present study in an attempt to improve productivity as: - PBI triploids are believed to have increased heterozygosisty over PBII triploids and diploids, and possibly better growth performance, and - Stocking of ponds with spawnings that have received shocks to prevent PBI extrusion would result in a greater proportion of females to males (as the frequency of induction is always 100%), therefore resulting in improved farm profit based on greater total harvestable product in terms of weight, and the fact that females attract higher prices per kilogram due to their larger size class. IONIZING RADIATION Ionizing radiation (IR) has been successfully used to prevent reproduction in a wide range of organisms and can be effective at sterilizing 100% of treated individuals. IR also plays a role in producing monosex populations through gynogenesis and androgensis in a range of aquatic species. Despite this, there is no information on the effects of IR on penaeid shrimp reproduction. Experiments in the present study assessed the capacity of IR to confer sexual sterilization in female and male M. japonicus. Initial experiments were focused on optimizing IR dose ranges. For these experiments, harvest age shrimp were treated with 0, 10 or 20 gray (Gy) of IR and their ability to produce viable offspring was assessed by performing rigorous reproductive evaluations. Later experiments assessed the reproductive performance of 10 month old M. japonicus treated with IR at postlarval stage 15 (PL15) (15 days since metamorphosis from mysis to postlarval stage 1). Initially, a lethal dose curve of IR for PL15 M. japonicus was established. Based on these results, PL15 shrimp were treated with IR at 0, 10, 15, 20, 25 and 30 Gy to examine the effect of IR dose on their reproductive capacity at 10 months of age. IR was found to impair the reproductive performance of M. japonicus when treated at harvest age and when treated as postlarvae and reared to reproductive maturity. IR was, however, not 100% effective at preventing the production of viable offspring at the reported doses. Harvest age male shrimp were more sensitive to IR, being reproductively impaired at 10 Gy as compared to 20 Gy for harvest age females. PL15 females treated with 0 Gy of IR matured and spawned more frequently than PL15 females treated with 10, 15 and 20 Gy of IR. There were, however, no other significant effects of IR observed on M. japonicus reproductive performance when treated at PL15. IR doses higher than 35 Gy resulted in 100% mortality (i.e. lethal dose rate) of PL15 M. japonicus within 30 days after treatment, whilst doses of 25 and 30 Gy significantly reduced postlarval survival compared to controls. From preliminary research, dose rates of 25 Gy or more resulted in 100% mortality of harvest age females, whilst 20 Gy significantly reduced harvest age male survival. These findings indicate that IR doses that do not result in 100% mortality of harvest age and postlarval M. japonicus can reduce their reproductive capacity, however, these doses of IR can not confer 100% sterility. GENETIC ENGINEERING The combined use of molecular biotechnology and genetics to investigate mechanisms of gender and fertility determination in numerous animal phyla has gained increased research attention over the last decade. However, to-date there is only one known penaeid shrimp gene for which the complete coding sequence has been isolated (but not functionally characterized) that is known to be involved in gender and fertility determination of other species. The present study assessed the potential of using genetic engineering methodologies to control gender and fertility of M. japonicus using a multi-step approach. Initially this study attempted to identify one or more candidate genes in M. japonicus that had been previously selected from a comprehensive literature search (sex-lethal, transformer, double-sex and vasa), and subsequently characterize their complete coding sequence. Expression profiles of the isolated genes were then determined throughout embryonic, larval and postlarval development, and in female and male gonad tissue to identify key periods during early development when their expression is naturally up-regulated. Using an RNAi approach, this study then attempted to silence the candidate genes from being expressed during these key developmental periods and assess the resulting loss-of-function genotypes. In addition to these experiments, this study also investigated the suitability of different real-time reverse transcription PCR (qRT-PCR) assay methodologies for the studied diverse morphological sample set (relative vs absolute quantitation) and investigated different methodologies to deliver nucleic acids into shrimp embryos (electroporation vs a polyethylenimine (PEI) transfection reagent). A partial coding sequence for a dsx-like gene homologue, DMRT2, and a complete coding sequence for a PL10 vasa-like gene was isolated from M. japonicus. The PL10 vasa-like gene was named Mjpl10. Both genes were differentially expressed during embryonic, larval and postlarval development, and in female and male gonad. Using absolute qRT-PCR, I demonstrated that dsx-like gene transcripts were present at low levels throughout the first 5 h of embryogenesis after which transcript abundance increased significantly by the time embryos were 6 h old, suggesting a functional role for dsx transcripts at this developmental stage. Expression of the dsx-like gene was consistently high but variable throughout the remainder of embryogenesis and during larval and postlarval development up until PL48 at which point dsx transcripts were no longer detectible. This expression pattern indicated a functional role for the isolated dsx-like gene from 6 h post-spawning until PL30. Transcripts of the dsx-like gene were also detected in testes at low levels and were close to undetectable in the ovary of mature adults. Using absolute qRT- PCR I also demonstrated that Mjpl10 transcripts were present in the first developmental stage sampled (2-cell embryos) onwards, suggesting it is maternally expressed, and continually at low levels throughout embryogenesis. Mjpl10 expression increased significantly in the first 25 h after hatching (nauplii IV) and then decreased in a linear fashion by 316-fold over the next 52 day period. Its continued expression throughout embryonic and larval development is compatible with a conserved role in early germ cell specification. Transcript levels of Mjpl10 were also detected in the ovary and testes of mature adults. To silence expression of the identified candidate genes in vivo, short hairpin RNA (shRNA) DNA expression vectors were constructed that were capable of in vivo transcription of a dsxlike and Mjpl10 shRNA molecule. These shRNA molecules would theoretically trigger the RNAi pathway and result in gene-specific silencing of the target dsx-like gene and Mjpl10 gene transcripts. The white spot syndrome virus immediate early 1 (WSSV IE1) promoter was chosen to drive expression of the shRNA molecules. The WSSV IE1 promoter was initially isolated and cloned into a vector to drive expression of a luciferase mRNA. WSSV IE1 promoter function was found to be >56% as efficient as the Orgyia pseudotsugata (Douglasfir Tussock moth) multicapsid nucleopolyhedrosis virus immediately-early 2 (OpIE2) promoter when compared in Fall Army worm, Spodopterea frugiperda (Sf) 9 insect cell culture. The vector containing the WSSV IE1 promoter was subsequently used to construct three recombinant DNA vectors capable of in vivo expression of a shRNA fragment of either the dsx-like gene, Mjpl10 or a placebo control luciferase gene. Three additional recombinant vectors were constructed with each containing a fragment of either the dsx-like gene, Mjpl10 or luciferase gene (identical to the sense strand of the shRNA fragments) that was inserted behind coding sequence for a green fluorescent protein and before the polyA tail. These three GFP fusion vectors were co-transfected with their corresponding shRNA expression vectors in Sf 9 cell culture. Theoretically mRNAs expressed by the GFP fusion vectors would be silenced and unable to express GFP if the corresponding shRNA expression vector was transcribing shRNA molecules that were capable of cleaving the GFP fusion mRNA polyA tail off the molecule through the RNAi pathway. Using this approach all three shRNA expression vectors constructed in this study were shown to be functional and capable of inducing a gene-specific silencing response in Sf 9 insect cell culture. Relative to a positive control, treatments transfected with the dsx-like shRNA expression vector had a reduced percentage of insect cells expressing GFP (i.e.18.07 ± 0.38% and 8.75 ± 0.15% compared to 100% in two independent experiments). Treatments transfected with the Mjpl10 shRNA expression vector also had a reduced percentage of cells expressing GFP relative to a positive control (i.e. 14.63 ± 0.59% and 9.19 ± 0.02% compared to 100% in two independent experiments). Similarly, treatments transfected with the luciferase shRNA expression vector had a reduced percentage of cells expressing GFP relative to a positive control (1.64 ± 0.02 and 0.47 ± 0.06% compared to 100% in two independent experiments). From these experiments it was evident that the WSSV IE1 promoter was functional, and that the shRNA fragments of the recombinant vectors were being expressed, folding and initiating a specific RNAi response. The three shRNA expressing recombinant DNA vectors and the positive transfection control luciferase mRNA expression vector were transfected into 1-cell M. japonicus embryos using the commercially available jetPEITM transfection reagent. Using qRT-PCR I demonstrated that jetPEITM DNA complexes were successfully transfected into 1-cell embryos as evidenced by the presence of in vivo transcribed exogenous luciferase mRNA in the positive transfection controls. Using qRT-PCR I then demonstrated that the Mjpl10 shRNA molecules were able to induce a small, but significant non-specific gene silencing response in vivo. The number of Mjpl10 mRNA copies detected per ng of total RNA was 14,108 ± 1,763 in the control compared to 6,701 ± 494 in the placebo shRNA treatment and 8,151 ± 1,151 in the genespecific Mjpl10 shRNA treatment. In comparison, the dsx-like shRNA molecules did not result in a non-specific silencing response. No specific gene silencing response was observed for either of the shRNA expression vectors. Given that the Mjpl10 and luciferase shRNA expression vectors were capable of reducing the total number of Mjpl10 mRNAs but no specific gene silencing was observed; the fact that the two endogenous targets, the dsx-like homologue and Mjpl10, are known to be expressed in specific cell types (i.e. the genital ridge and the germ cell lineage respectively) and a measurable gene silencing result would therefore require that large numbers of shRNA be in vivo transcribed in these specific cell types; and that the shRNA expression vectors had been demonstrated to be functional in Sf 9 cell culture, these results indicate that the WSSV IE1 promoter was not strong enough to drive in vivo expression of suitable quantities of shRNA molecules to induce a measurable gene silencing response. Future research should isolate alternative shrimp specific constitutive promoters such as β-actin which are highly active at all life-history stages in the majority of cell types. The use of such promoters instead of the WSSV IE1 promoter would ensure a high level of expression of recombinant DNA vectors that are transfected in vivo. Comparison of the relative and absolute qRT-PCR quantitation approaches revealed significant differences in transcript level profiles between the two procedures for both target genes, the dsx-like gene and Mjpl10. When 18S rRNA was used as a reference, target gene expression was more similar to that of the absolute method than when β-actin was used as a reference. Variability between the relative and absolute procedures occurred for a greater Percentage of the embryonic stages compared to later developmental stages. This study indicates that the use of 18S rRNA or β-actin as reference genes for studying gene expression patterns in M. japonicus embryonic, larval, postlarval and gonad samples will give significantly variable results, and illustrates the proposition that housekeeping genes are not necessarily appropriate references for qRT- PCR data normalization. Until suitable reference genes are characterized, gene expression experiments using the studied M. japonicus tissues of different morphological developmental stages should use absolute quantification procedures. Finally, comparison of electroporation and the polyethylenimine (PEI) transfection reagent procedures demonstrated the jetPEITM transfection reagent was the most suitable means by which to transform 1-cell M. japonicus embryos. Electroporation was not found to be suitable for delivering nucleic acids into 1-cell M. japonicus embryos at quantities greater than what is detectible from nucleic acids attached to the outer embryo surface after DNase or RNase treatment. However, the commercially available jetPEITM transfection reagent was able to deliver nucleic acids into 1-cell M. japonicus embryos when soaked in the transfection media for between 50 min to 4 h post-spawning. Transfection was most successful when complexation was performed for 15 min in a sodium chloride buffer, followed by a 3 to 4 h incubation in sterile seawater prior to the addition of embryos. Greatest transformation rates (as measured by the number of exogenous mRNA template copies per ng of total RNA) were achieved when 1 mL of seawater and embryos were added to complexation mixtures within the first 5 min of spawning.